Compositions for the treatment of cancer, and methods for testing and using the same

ABSTRACT

A composition comprising leukotoxin proteins isolated from a bacterium is provided. In this composition, greater than 85% of the leukotoxin proteins are chemically modified at a basic amino acid residue, and the proteins induce cell death in myeloid leukocytes, while remaining substantially non-toxic to lymphoid leukocytes, lymphocytes, and red blood cells. Also provided is a method of selectively inducing cell death in myeloid leukocytes. The method comprises contacting the myeloid leukocytes with a composition comprising leukotoxin proteins. These leukotoxin proteins may be isolated from the NJ4500 strain of  Actinobacillus actinomycetemcomitans.  A method of purifying leukotoxin protein from the NJ4500 strain of  Actinobacillus actinomycetemcomitans  is also provided, as well as an assay that allows for the rapid determination of the activity of a given drug against leukemic cells either taken from a patient or derived from a cell line. The assay is performed in the presence of whole blood or serum.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation In Part of PCT Application No.PCT/US2006/045258, filed Nov. 25, 2006, which, in turn, claims priorityunder 35 U.S.C. §119(e) from U.S. Provisional Application Ser. No.60/739,537, filed Nov. 25, 2005; and co-pending U.S. Non-Provisionalapplication Ser. No. 12/150,038, filed Apr. 23, 2008, which, in turn,claims priority under 35 U.S.C.§119(e) from U.S. Provisional ApplicationSer. No. 60/925,794, filed Apr. 25, 2007. The entire contents of allprior listed applications are incorporated by reference herein.

RELATED FEDERALLY SPONSORED RESEARCH

The work described in this application was sponsored at least in part,under Grant No. R01 DE16133, from the National Institute of Dental andCraniofacial Research, and under Grant No. NIH R01DE16133, from theNational Institutes of Health. Accordingly, the Government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention is generally related to agents and compositionsthat demonstrate the ability to treat certain cancers, and to a methodand system for the testing of such agents and compositions underphysiological conditions. More particularly, the agents and compositionscomprise a repeat in toxin (RTX) molecule that demonstrates leukocytespecificity, and that specifically targets myeloid leukocyte cells, aswell as to an assay that allows for the rapid determination of theactivity of a given drug against cancer cells, such as leukemic cells,either taken from a patient or derived from a cell line.

BACKGROUND OF THE INVENTION

Bacteria and their toxins have been investigated for their anticanceractivities. In the 1970s, bacteria (such as non-pathogenic Clostridium)were used for the treatment of malignant brain tumors, but the tumorsrecurred in these brain tumor patients. More than 100 microorganismshave been studied for their potential anticancer activities, and manybacteria have growth specificity for tumors that is 1000 times greaterthan for other tissue.

While their anti-tumor activities make many bacteria attractivetherapeutic agents, there are inherent risks to administering livebacteria to humans. A safer and more effective strategy has been to usebiological toxins, specifically from bacteria, as therapeutic agents.Bacterial toxins are not only toxic, but are also highly specific forcertain cell types, or can be engineered to be specific by fusing thetoxin to other molecules. Many bacterial toxins are able to entermammalian cells where they exert their toxic effects. Because ofextensive evolutionary adaptation between bacteria and their hosts,bacteria have become very good at “developing” highly effective toxins.

Each year, more than 60,500 people die of hematologic malignancies(leukemia, lymphoma, myeloma) with more than 110,000 new annualdiagnoses in the US alone. Current treatment for these cancers includesthe use of synthetic compounds that target the cell division process ofnearly all cells of the body, not just the cancerous ones. As a result,devastating side effects are all too common. Furthermore, a significantpercentage of patients eventually show resistance to many of the drugs,thus rendering treatment largely ineffective. Indeed, there is an effortto identify agents that induce cancer cell death by methods other thandamage to DNA or cell division.

While the drugs currently in use are toxic for cells, they are nothighly specific. A new class of therapeutic agents for the treatment ofhematologic malignancies, and cancer in general, includes drugs thatexhibit specificity for predominantly the cancerous cell type. Examplesof targeted therapeutics include Rituximab, which is a monoclonalantibody against B-lymphocytes, and Mylotarg, an antibody-anti-tumorantibiotic fusion directed against cells of myelomonocytic lineage.

Actinobacillus actinomycetemcomitans is a Gram negative pathogen thatinhabits the oral cavities of humans. A. actinomycetemcomitans is theetiologic agent of localized aggressive periodontitis (LAP), a rapidlyprogressing and destructive disease of the gingiva and periodontalligaments. Among its many virulence factors, A. actinomycetemcomitansproduces an RTX (repeats in toxin) leukotoxin. A. actinomycetemcomitansleukotoxin is an approximately 115 kDa protein that kills specificallyleukocytes of humans and Old World Primates. Leukotoxin is part of theRTX family that includes E. coli α-hemolysin (HlyA) and Bordetellapertussis adenylate cyclase (CyaA). Leukotoxin may play an importantrole in A. actinomycetemcomitans pathogenesis by helping the bacteriumdestroy gingival crevice polymorphonuclear leukocytes (PMNs) andmonocytes, resulting in the suppression of local immune defenses.

The initial identification and testing of novel anti-cancer agentsrelies on in vitro killing assays using relevant cancer cell lines.While in vitro assays performed under cell culture conditions proveuseful and necessary for preclinical testing of new therapeutics,extrapolation to the physiological conditions of a living organism isoften difficult or impossible (27). Because of the high cost of drugdevelopment ($800 million), new drug screens are constantly being soughtto more efficiently eliminate or identify candidate therapeutic agents(27). Indeed, increasing the clinical success rate from ⅕ to ⅓ becauseof more effective preclinical drug screens would reduce drug developmentcosts by more than $200 million (27).

The activity, specificity, or toxicity of a compound in thephysiological environment can vary significantly from cell cultureconditions. While no in vitro assay or screen can represent thecomplexity of the human body, several assays have been developed to moreclosely mimic in vivo conditions. Several of these assays include thecolony forming cell assay using bone marrow cells (27,29), hepatic drugbiotransformation assays (3), and assays in whole blood (4,45). Becausemost chemotherapeutic agents are administered intravenously and aretherefore immediately affected by blood cell components, screening forpotential drugs in the presence of whole blood would be expected toyield more meaningful results. Blood contains biological components,such as proteases, antibody, and blood cells, which can affect thenature of a compound. For example, red blood cells and plasma proteinsare known to affect the pharmacokinetics of drugs such as theanti-cancer agents docetaxel and gemcitabine (8,9). Vaidyanathan et al.(43) also reported that the cardioprotective drug, dexrazoxane, inhibitsbinding of the anti-cancer agent, doxorubicin, to red blood cells andthat this interaction alters the pharmacokinetics of doxorubicin, andClarke et al. (4) used an in vitro whole blood assay to study thebinding affinity of a surrogate anti-CD 11 a monoclonal antibody toblood components. In addition, leukocytes produce a cytochrome P450isoform (CYP2E1) that is involved in drug biotransformation (3). Thus,identifying and studying drugs in the presence of whole blood or bloodcomponents can offer a unique advantage over assays using cells inmonoculture.

For studies on leukemia therapeutics, the cell line HL-60 is used as astandard target cell line. HL-60 cells were originally isolated from a36-year-old female patient with acute promyelocytic leukemia (13).Testing the efficacy of anti-leukemia therapeutics against HL-60 cellsin whole blood or other biological material is currently a challenge dueto the inefficiency in differentiating the viability of HL-60 cells fromother cells.

Thus, there remains a need for the identification and development oftherapeutic agents and strategies, for the treatment of cancers such asleukemia, and for the development of effective testing methodology, suchas efficient screens for therapeutics such as anti-leukemia agents, andparticularly, for the facilitation of preclinical studies on a highlyspecific bacterial leukotoxin as a novel anti-leukemia therapeuticagent.

SUMMARY OF THE INVENTION

Accordingly, in a first embodiment of the invention, a composition forthe treatment cancers, and particularly, hematologically relatedcancers, is disclosed that comprises leukotoxin proteins isolated from abacterium. In this composition, greater than 85% of the leukotoxinproteins are chemically modified at a basic amino acid residue, and theproteins induce cell death in myeloid leukocytes, while remainingsubstantially non-toxic to lymphoid leukocytes, lymphocytes, and redblood cells.

Also, in a method of treatment aspect, there is provided a method ofselectively inducing cell death in myeloid leukocytes. The methodcomprises contacting the myeloid leukocytes with a compositioncomprising leukotoxin proteins. These leukotoxin proteins may beisolated from the NJ4500 strain of Actinobacillus actinomycetemcomitans.A method of purifying leukotoxin protein from the NJ4500 strain ofActinobacillus actinomycetemcomitans is also provided.

Accordingly, in a second embodiment of the invention, a stablebioluminescent HL-60 cell line whose viability can be rapidly andeffectively determined in the presence of whole blood and live animalshas now been developed along with an assay that allows for the rapiddetermination of the activity of a given drug against a cell sample,such as leukemic cells, either taken from a patient or derived from acell line. The assay is carried out in the presence of whole blood orserum. This quantitative assay can screen thousands of drugs at a timeor multiple concentrations of a drug in a 96- or 384-well format.

The present assay uses HL-60 cells that have been engineered that stablyexpress firefly luciferase and produce light, whereby suchbioluminescent HL-60luc cells may be rapidly detected in whole blood,eg. with a sensitivity of approximately 1000 viable cells. Asdemonstrated herein, treatment of HL-60luc cells with a bacterialleukocyte-specific toxin or the drug chlorambucil reveals that thebioluminescent viability assay is more sensitive than the trypan bluedye exclusion assay. HL-60luc cells administered intraperitoneally (i.p)or intravenously (i.v.) were visualized in living mice using an in vivoimaging system (IVIS). The rapidity and ease of detecting HL-60luc cellsin biological fluid indicates that this cell line can be used in highthroughput screens for the identification of drugs with anti-leukemiaactivity under physiological conditions.

Accordingly, it is a principal object of the invention to provide aseries of modified leukotoxin proteins, and compositions comprisingthem, that can be used for the treatment of cancers, ad particularly,cancers that are hematologically related.

It is a further object of the invention to provide a method for thepreparation of the modified leukotoxin proteins by their isolation andpurification from a bacterium, eg. from the NJ4500 strain ofActinobacillus actinomycetemcomitans.

It is a yet further principal object of the invention to provide a anassay and associated methodology, for the testing of candidatetherapeutic agents for the treatment of cancers, that is rapid andefficient, and that can assess the activity of the candidate agentsunder physiological conditions.

A still further object of this invention is to provide methods oftreating cancers and conditions by the administration of the leukotoxinproteins and compositions comprising them.

Other important objects and features of the invention will be apparentfrom the following description of the invention taken in connection withthe accompanying drawings in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows fluorescence microscopy images of leukemia HL-60 cells whenexposed to LtxA.

FIG. 2 is a graph showing activity data of two forms of LtxA againsthuman red blood cells.

FIG. 3 is a series of images showing the effectiveness of NJ4500 LtxA invivo.

FIG. 4 a bar graph representation of data showing the sensitivity ofhuman red blood cells to the JP2 and NJ4500 forms of LtxA.

FIG. 5 is a bar graph representing data on the toxicity of the JP2 andNJ4500 forms of LtxA against HL-60 cells.

FIG. 6 is a graph of time vs. cell death for various cell types in wholehuman blood during incubation with 0.2 μg/ml NJ4500 LtxA.

FIG. 7 is a two-dimensional gel electrophoresis of two forms of LtxA.

FIGS. 8A and 8B show the construction of a stable luciferase-expressingHL-60 cell line wherein (A) HL-60 cells were transfected with pMP1 andthen grown in wells with different concentrations of geneticin.Bioluminescence was detected with the IVIS 50 instrument and (B) Growthcurves for parental HL-60 and engineered HL-60luc cells. Cells weregrown in RPMI as described and viable cells were counted with the ViCELLcell counter.

FIGS. 9A-9C shows the detection of HL-60luc cells in whole bloodincluding (A) Kinetics of BL over time. HL-60luc cells were mixed withblood and luciferin and then imaged with the IVIS 50 instrument at theindicated time points. The observed pattern was highly reproducible. (B)Detection limit of HL-60luc cells. Cells were mixed with blood andluciferin and then incubated for one hour before imaging. (C) The numberof HL-60luc cells shows a linear correlation with BL.

FIG. 10 shows effects of LtxA on cells. (A) Lysis of human red bloodcells by LtxA from two different strains of A. actinomycetemcomitans.(B) HL-60 and HL-60luc cells are equally sensitive to killing by LtxAfrom strain NJ4500. Assays were performed in RPMI medium and viabilitywas determined using the trypan blue dye exclusion assay.

FIGS. 11A-11C show the cytotoxicity of LtxA and chlorambucil. (A)Activity of LtxA against HL60luc cells in whole human blood and RPMImedium. Viability was measured using BL. (B) Comparison between BL andtrypan blue as viability assays for LtxA-mediated cytotoxicity. Cellswere incubated in RPMI medium with LtxA or buffer for 4 hours andviability was determined. (C) Comparison between BL and trypan blue asviability assays for chlorambucil-mediated cytotoxicity. Cells wereincubated in RPMI medium with chlorambucil or buffer for 24 hours andviability was determined

FIG. 12 shows bioluminescent imaging of HL-60luc cells in living mice.Swiss-Webster mice were anesthesized with XXX and injected with 106HL-60luc cells intraperitoneally (i.p.; top) or intravenously (i.v.;bottom) and followed by luciferin i.p. Mice were imaged with the IVIS 50instrument at different times post luciferin injection. The scale on theright of each image indicates surface radiance(photons/second/cm²/steradian).

DETAILED DESCRIPTION OF THE INVENTION

Leukotoxin is an effective cell-delivery protein, permeating leukemiacells and penetrating to the inside of specific cells. Leukotoxinmediated cell-delivery is demonstrated by introducing fluorescingmolecules to specific cells, and measuring cell-delivery by monitoringthe fluorescence by fluorescent microscopy. As shown in FIG. 1, theleukotoxin LtxA facilitates delivery of fluorescein into HL-60 leukemiacells. The leukotoxin forms pores or disruptions in the host cellmembranes, and these openings in the membrane may allow the passage andentry of small molecules. In FIG. 1, HL-60 cells were treated withfluorescein, a reagent that can be easily tracked by fluorescencemicroscopy. Fluorescein exhibits a green fluorescence color under themicroscope, and is approximately the same molecular weight as many ofthe cancer drugs currently in use. The cells treated with leukotoxin(LtxA) and fluorescein (FIG. 1, bottom panel) exhibited more intense andabundant fluorescence than the cells treated with fluorescein alone(FIG. 1, center panel), indicating that leukotoxin is able to increasethe number of fluorescein molecules that enter the cells.

Not only is leukotoxin capable of penetrating cells, but thispenetration is toxic and lethal to HL-60 cells. HL-60 cells weremodified to express luciferase genes, and with this HL-60luc system, itwas shown that at certain concentrations, leukotoxin is quite toxic tothe HL-60luc cells. By monitoring the luminescence of the cells, nearly80% of the cells were killed by concentrations of leukotoxin as low as200 ng/ml.

Data reflecting the sensitivity of HL-60luc cells to leukotoxin is shownin FIG. 2. The activity of purified leukotoxin against HL-60luc cells invitro is quantified. The leukotoxin used in this experiemenbt was LtxAisolated from the NJ4500 strain of Actinobacillus actinomycetemcomitans.The LtxA was mixed with HL-60luc cells at various concentrations asindicated, and incubated for two hours, and then imaged with the IVIS 50instrument. Relative viability was calculated by quantifying the numberof photons produced in each well. Significant cell death was observedafter two hours for concentrations of 2.0 μg/ml, 0.2 and 0.02 μg/ml.

To determine if the leukotoxin LtxA has activity in vivo, two SwissWebster mice were injected i.p. with 10⁶ HL-60luc cells. One of the micewas injected with 8 μg of LtxA i.p. immediately following HL-60luc cellinjection. Both mice then received an i.p. injection of luciferinsubstrate. The mice were monitored by in vivo bioluminescence imagingwith an IVIS 50 imaging system immediately following injection of theluciferin. The luminescent signal was visible and intense in the controlmousee that did not receive the LtxA injection. In contrast, the mousethat received LtxA showed essentially no luminescent signal, showingthat the LtxA had killed the HL-60luc cells in vivo.

Forms of LtxA include the JP2 form (isolated from the JP2 strain ofActinobacillus actinomycetemcomitans) and the NJ4500 form (isolated fromthe NJ4500 strain of Actinobacillus actinomycetemcomitans). NJ4500 LtxAis well tolerated by the Swiss Webster mice. Two mice, weighingapproximately 45 grams each, were injected with 10 μg of NJ4500 LtxAintravenously. These mice were monitored over a five-month period, andduring this period, the mice remained healthy, did not lose weight, andhad no apparent adverse reaction to the LtxA.

The individual forms of LtxA show different cell specificity. The NJ4500and JP2 forms of LtxA demonstrate specificity to different types ofblood cells, as demonstrated in FIG. 4. The JP2 form of LtxA is lethalto human red blood cells, whereas human red blood cells are insensitiveto the NJ4500 form of LtxA. Thus, the NJ4500 form of LtxA is lethal toHL-60 cells (as shown in FIG. 3), but innocuous to human red blood cells(as shown in FIG. 4). The JP2 form is quite lethal to human red bloodcells, but as shown in FIG. 5, is less deadly to leukemia cells than theNJ4500 form. The data displayed in FIG. 4 was collected by a trypan bluedye excusion assay. Leukotoxin protein LtxA (2 μg/ml) was added to 1×10⁶HL-60 cells, and were incubated for 90 minutes at 37° C. The cells weremeasured for viability with the trypan blue dye exclusion assay.

The activity of the JP2 and NJ4500 forms of LtxA against HL-60 cellsdiffers dramatically. In FIG. 5, a bar graph representing the toxicityof the two forms of LtxA against HL-60 cells. As shown by the bar graph,the NJ4500 form of LtxA is much more lethal to the leukemia cell linethan the JP2 form of the protein. Accordingly, not only is the NJ4500form of LtxA non-lethal to human red blood cells (unlike the JP2 strainof LtxA), but the NJ4500 strain of LtxA is more lethal to leukemia cellsthan the JP2 strain of LtxA, thus indicating that the NJ4500 form ofLtxA is a desirable leukemia or blood disease treatment as it is highlytoxic to leukemia cells, but not to human red blood cells. LtxA providesa highly specific approach to treat hematologic malignancies, such asleukemia, lymphoma, and myeloma, without damaging other blood cells,such as red blood cells.

The data displayed in FIG. 5 was collected using a trypan blue exclusionassay. HL-60 cells were mixed with 2 μg/ml final concentration of LtxAfrom JP2 and NJ4500 as indicated and incubated for 90 minutes at 37° C.Equal amounts of LtxA from either strain were mixed with approximately5×10⁶ cells/ml of HL-60 cells and incubated for ninety minutes. Celldeath was then assayed using the trypan blue dye exclusion assay. LtxAfrom NJ4500 was more effective at killing HL-60 cells than was LtxA fromJP2. The toxin from NJ4500 was approximately twice as active and thisresult was highly reproducible for even different preparations of LtxAover four different experiments.

The NJ4500 strain of LtxA is also active in whole human blood. Wholehuman blood was mixed with LtxA (2.0 μg/ml final conc.) and incubatedfor 4 hours at 37° C. The mixtures were then mixed with red blood cell(RBC) lysis buffer (eBioscience) and the RBCs were lysed according tothe manufacturer's protocol. The remaining white blood cells (WBCs) werethen resuspended in PBS and cells were counted using a ViCell counter(Beckman Coulter), which employs the trypan blue dye assay to measureviability. The sample that was not treated with LtxA had 93% viability,while the sample that was treated with LtxA had a viability of 42%.Because the RBC's were lysed and removed before viability was measured,the viability measurement assesses only the viability of the remainingwhite blood cells. The NJ4500 LtxA caused death in nearly 60% of thewhite blood cells.

NJ4500 LtxA displays unique sensitivity among the blood cells found inwhole blood. At a concentration of 20 μg/ml, LtxA from NJ4500 showed ahigh level of specificity in inducing cell death in human whole blood.After four hours incubation at 20 μg/ml LtxA, the red blood cells,basophils, and lymphocytes suffered no significant cell death. Incontrast, the 60% of the white blood cells were killed, andapproximately 95% of the neutrophils, monocytes, and eosinophils werekilled by the LtxA.

Similar specificity was shown over time at lower doses of NJ4500 LtxA.FIG. 6 is a graph representing data that was collected over time, andshows that NJ4500 LtxA specifically target certain blood cell types.Neutrophil cells appear most sensitive to the relatively lowconcentration of 0.2 μg/ml LtxA with nearly 80% of neutrophils killed inonly one hour. Both basophils and white blood cells (or leukocytes) werekilled rapidly by the NJ4500 with about 60% of the cells dying within anhour. Monocytes are quite sensitive to NJ4500 LtxA, as nearly 100% ofthe monocytes died, however significant amounts of cell death requiredlonger incubation periods. In contrast, red blood cells (erthrocytes)and lymphcytes were completely insensitive to 0.2 μg/ml NJ4500 LtxA overtime.

All histological data presented herein was collected through histologyexaminations. The blood samples were smeared onto a glass slide and thenprocessed and stained on a Coulter LH slide maker, using a Wright stainfor differentials.

The JP2 and NJ4500 forms of LtxA isolated Actinobacillusactinomycetemcomitans differ functionally in that the NJ4500 showsgreater toxicity towards leukemia cells, along with greater specificity.The NJ4500 form of LtxA is highly specific towards white blood cells(leukocytes). Specifically, the NJ4500 form of LtxA is highly specifictowards basophils, neutrophils, and monocytes. The NJ4500 form of LtxAis also highly specific towards eosinophils. This form does not inducesignificant cell death in red blood cells (erythrocytes) or lymphocytes.These functional distinctions may be related to structuralmodifications. Specifically, NJ4500 LtxA is highly modified with fattyacids.

A two-dimensional gel electrophoresis of JP2 and NJ4500 LtxA is shown inFIG. 7. The gel shown in FIG. 7 shows that the JP2 form of LtxA containsa significant amount of protein with an isoelectric point of at least9.0. To create the 2-D gel, LtxA (20 mg) from JP2 or NJ4500 wasseparated first by isoelectric point through a pH gradient of 7-10. LtxAseparated by isoelectric focusing was then separated by mass usingpolyacrylamide gel electrophoresis. Protein was visualized with SYPROruby stain. The LtxA samples were prepared for 2-D gel electrophoresisby processing 20 mg with a 2-D gel clean-up kit according to themanufacturer's directions (Amersham Biosciences, Piscataway, N.J.).Following the clean-up, the pelleted protein was resuspended in 182 mlrehydration buffer (Bio-Rad, Hercules, Calif.) and mixed with 3.7 ml DTT(500 mg/ml) and 2 ml 1% bromophenol blue. The sample was then loadedonto a pH 7-10 IPG strip and processed for isoelectric focusingaccording to the manufacturer's protocol (Bio-Rad, Hercules, Calif.).After isoelectric focusing, the IPG strip was layered atop a 10%polyacrylamide gel and resolved in SDS buffer for several hours.Visualization of protein spots was accomplished by staining the gel inSYPRO ruby protein stain (Bio-Rad, Hercules, Calif.).

Two other RTX toxins, E. coli HlyA and B. pertussis CyaA, are modifiedcovalently with fatty acid moieties at internal lysine residues. In E.coli, hlyC is a fatty acyl transferase and is required for modificationof HlyA. Based on the presence of an hlyC homologue in A.actinomycetemcomitans (ltxC), it is predicted that A.actinomycetemcomitans LtxA is also modified. Modification of HlyA andCyaA is required for toxin activity and the degree of modification isdirectly correlated with toxicity.

The two forms of LtxA were subjected to two-dimensional gelelectrophoresis to assess whether differential modification of theproteins accounted for the functional differernces. The two-dimensionalgel electrophoresis showed that LtxA from both strains exists inmultiple isoforms (See FIG. 7). Several representative spots wereexcised and subjected to trypsin digestion and MALDI-TOF MS analysis.MALDI-TOF MS confirmed that all species were A. actinomycetemcomitansLtxA (data not shown).

The predicted pI of LtxA based on primary amino acid sequence isapproximately 9. (Represented by the rightmost spot and small arrow inFIG. 7). Modification of lysine residues with fatty acids shifts the pItowards the acidic end, therefore, a greater fraction of the NJ4500 LtxAis modified compared to LtxA from JP2.

Approximately half of the JP2 LtxA is completely unmodified (asrepresented by the dense spot at approximately pI 9.). In contrast, noneof the NJ4500 LtxA appears to be completely unmodified in the 2-D gelshown in FIG. 7.

Other RTX toxins are modified with fatty acids and this modification isrequired for activity. Modification of RTX toxins may contribute to hostand cell type specificity. E. coli, a-hemolysin, is modified at twointernal lysine residues with C14, C15, or C17 fatty acid residues.Because E. coli can incorporate into HlyA three different fatty acids attwo different lysine residues, preparations of HlyA are heterogeneous.Based on the two-dimensional gel electrophoresis data shown in FIG. 7,LtxA is even more heterogeneous than HlyA.

LtxA has approximately 100 lysine residues and modification at severalof them with different types of fatty acids accounts for the relativelylarge number of different isoforms. LtxA purified from NJ4500 is moreactive than LtxA from the JP2 strain. Nearly all of NJ4500 LtxA existedin some type of modified form whereas preparations of JP2 LtxA containeda significant amount of unmodified form (FIG. 7). Consistent with otherRTX toxins, the unmodified toxin from JP2 is inactive against HL-60cells. Thus, the percent of active LtxA molecules in preparations fromNJ4500 is greater than from JP2.

A fatty acid is defined as a long-chain monobasic organic acid or ahydrocarbon chain. Fatty acids associated with NJ4500 LtxA were analyzedby Mylnefield Research Services, Lt., the Lipid Analysis Unit, Scotland.The protocol for analysis of myristoylated proteins described byNeubert, T. A. and Johnson, R. S. (Methods Enzymol., 250, 487-494(1995)) was followed. It involves acidic hydrolysis of the protein,followed by acid catalysed methylation of the fatty acids for analysisby gas-chromatography. Approximately 3 mg of LtxA was subjected to thisprotocol, and preliminary data shows that at least myristic acid (C14)and palmitic acid (C16) are present in the LtxA protein.

The pI values of the two-dimensional gel indicated that basic residuesare modified (making the protein more acidic). Any basic residue may bechemically modified by a fatty acid to reduce the pI. Because it isknown that the lysine residues of RTX family members are modified byfatty acids, and that such a chemical modification increases activity ofthe RTX familiy members, the LtxA protein is likely to be modified atlysine residues.

Based on the data collected to date, the specificity and increasedactivity evident in the NJ4500 form of LtxA can be attributed to thecomposition of leukotoxin proteins with greater than 85% of theleukotoxin proteins chemically modified at a basic amino acid residue.This composition is an LtxA composition isolated from a bacterium suchas Actinobacillus actinomycetemcomitans, and preferably the NJ4500strain of Actinobacillus actinomycetemcomitans.

This composition as described induces cell death in myeloid leukocytes.The composition is specific to any of white blood cells, neutrophils,monocytes, basophils, and eosinophils. Myeloid cells are cells belongingto the white blood cell lineage, and consist of granulocytes (basophils,neutrophils, eosinophils), monocytes, erythrocytes and platelets. TheLtxA composition is specific as it is substantially non-toxic tolymphoid leukocytes.

White blood cells are a type of cell formed in the myelopoietic,lymphoid, and reticular portions of the reticuloendothelial system.Lymphocytes are a white blood cell formed in the lymphatic tissuethroughout the body (eg. Lymph nodes, spleen, thymus, tonsils, Peyerpatches, and sometimes in the bone marrow). In normal adultsapproximately 22-28% of the total number of white blood cells in thecirculating blood are lymphocytes.

The specificity of the NJ4500 LtxA is especially useful as a treatmentagainst acute myeloid leukemia (AML) and chronic myeloid leukemia (CML)because these are diseases in which only the myeloid cells aremalignant. Thus, LtxA provides a high level of toxicity to certainmyeloid cells, while leaving the red blood cell population unharmed, asthe NJ4500 is substantially non-toxic to lymphocytes and red bloodcells.

As discussed above, the NJ4500 LtxA composition of the inventionincludes chemical modifications, and the chemical modifications includefatty acid modifications to basic amino acid residues. Preferably, thebasic amino acid residue that is modified is a lysine residue, andgreater than 90% of the leukotoxin proteins are chemically modified atat least one basic amino acid residue.

Also discussed above, the NJ4500 LtxA compositions of the invention havea pI less than 9. Within the composition, 85% of the leukotoxin proteinshave a pI less than 9.0. In another embodiment, 90% of the leukotoxinproteins have a pI less than 9.0, and in yet another embodiment, 95% ofthe leukotoxin proteins have a pI less than 9.0. In still anotherembodiment, 100% of the leukotoxin proteins have a pI less than 9.0.

As shown in FIG. 7, most of the NJ4500 LtxA proteins have a pI less than8.5. In one embodiment of the invention, 85% of the leukotoxin proteinshave a pI less than 8.5, and in another embodiment, 90% of theleukotoxin proteins have a pI less than 8.5. The invention includescompositions where 95% of the leukotoxin proteins have a pI less than8.5, and in yet another embodiment, 100% of the leukotoxin proteins havea pI less than 8.5. The leukotoxin proteins are isolated fromActinobacillus actinomycetemcomitans, and in another embodiments, theleukotoxin proteins are isolated from the NJ4500 strain ofActinobacillus actinomycetemcomitans.

Because NJ4500 demonstrates a unique specificity among RTX familymembers, one embodiment of the present invention includes an RTX familyprotein that selectively lyses white blood cells more effectively thanred blood cells. The RTX family member is substantially non-toxic to redblood cells.

Also provided is a pharmaceutical composition comprising leukotoxinproteins and a pharmaceutically acceptable carrier, along with a methodof selectively inducing cell death in myeloid leukocytes comprisingcontacting the myeloid leukocytes with a composition comprisingleukotoxin proteins.

In another embodiment, a method of killing a target cell by contactingthe target cell with leukotoxin proteins is provided. In this methodeach leukotoxin protein has a pI less than 9.0. In one embodiment ofthis method, the myeloid leukocyte cells die at a faster rate thanlymphoid cells.

In still another embodiment, a method of treating a blood disease isprovided. This method comprises administering a composition ofleukotoxin proteins isolated from the NJ4500 strain of Actinobacillusactinomycetemcomitans to a subject suffering from the blood disease. Intreating a blood disease, a chemotherapeutic pharmaceutical may beadministered to the subject in conjuction with the leukotoxin. Someappropriate chemotherapeutic pharmaceuticals include idarubicin,cytarabine, etosposide, daunorubicin, mitoxantrone, and melphalan. Othercommon chemotherapeutic agents for the treatment of leukemia andlymphoma include Chlorambucil, Fludarabine phosphate, Cytarabine, andDaunorubicin hydrochloride. These drugs share the common property ofbeing highly toxic to humans, affecting many different tissue and organsystems of the body. Bone marrow suppression, severe neurologic effects,infertility, pulmonary, and gastrointestinal effects are some of theadverse effects exhibited by these drugs. Many of the drugs act byinhibiting DNA synthesis, a process that all dividing cells carry out.Most cells of the body are targeted by these non-specificpharmaceuticals. Any suitable pharmaceutical agent may be used inconjunction with LtxA, and the combination of a pharmaceutical agentwith leukotoxin is intended to reduce the dose of the pharmaceuticalnecessary to achieve effective results in patients.

In addition to the potential uses as an anti-cancer agent,Actinobacillus actinomycetemcomitans leukotoxin may serve as a potentanti-viral. Specifically, HIV replicates and resides inside macrophagesand T-lymphocytes. Viruses are difficult to combat because they often“hide” from the immune system inside host cells. Leukotoxin coulddestroy those macrophages that are infected with HIV, allowing the virusto be released and attacked by the natural host immune defenses. Thistreatment would be different in that the therapy would not be directedagainst the virus (which would select for resistant HIV mutants), butrather against the host cell in which the virus resides.

In one embodiment the blood disease is leukemia, lymphoma, or myeloma,and in another embodiment, leukotoxin is used in a method of selectivelysensitizing myeloid leukocyte cells to permeates. This method comprisescontacting the myeloid leukocyte cells with a composition comprisingleukotoxin proteins, wherein each leukotoxin protein has a pI less than9.0, and lymphoid cells are substantially unsensitized to permeates bythe composition.

In another embodiment, a method of purifying a RTX family protein fromActinobacillus actinomycetemcomitans comprises:

-   -   a. inoculating a single colony of Actinobacillus        actinomycetemcomitans into a fresh broth and growing cultures;    -   b. adding the growing cultures to fresh broth, adding beads and        incubating;    -   c. centrifuging the incubated culture, forming a pellet and a        supernatant;    -   d. filtering the supernatant through a membrane to provided a        filtered supernantant:    -   e. mixing (NH₄)₂SO₄ and the filtered supernatant together to        form a mixture;    -   f. centrifuging the mixture to form a mixture pellet;    -   g. resuspending the mixture pellet in buffer to form a protein        resuspension;    -   h. passing the protein resuspension through a column; and    -   i. collecting the protein eluting off the column.

In a further aspect of the invention, an assay system and method arepresented, that enables the testing of anti-cancer drugs such as theleukotoxins of the present invention, under physiological conditions,with the consequence and benefit that a relevant prediction of activityis rapidly and efficiently obtained.

Screens for compounds and proteins with anti-cancer activity employviability assays using relevant cancer cell lines. For leukemia studies,the human leukemia cell line, HL-60, is often used as a model system. Tofacilitate the discovery and investigation of anti-leukemia therapeuticsunder physiological conditions, and in accordance with this invention, abioluminescent HL-60 cell line has been engineered that can bevisualized in whole human blood and living mice and whose viability canbe rapidly determined. A WBC-specific bacterial toxin has been shown tobe active in blood. The engineered HL-60luc cells of the inventionbehave similar to the parental HL60 cell line. When used in abioluminescence imaging assay (BLI) as discussed in detail herein, theBLI signal peaked approximately one hour following the addition ofluciferin but remained relatively high for several hours. This type ofin vitro kinetics where an early peak in luminescence is followed by aslow decline is consistent with other BLI cell lines. The detectionlimit of 1000 viable cells is also consistent with other reports(35,36). Because human blood contains plasma proteins, such as antibodyand proteases, and other cells, that may affect the activity,availability, or stability of a drug, the anti-leukemia assays withHL-60luc cells in the presence of blood can yield more physiologicalresults than with buffer or growth media alone.

In vivo bioluminescence imaging (BLI) is a technology that allowsvisualization of live bioluminescent cells (mammalian, bacterial,viruses) in complex biological material and living animals (24,31).Firefly luciferase has been used extensively in reporter systems and itsexpression can be measured quantitatively using a luminometer or highlysensitive charge coupled device (CCD) camera. Rocchetta et al. (32)found that the CCD camera was approximately 25 times more sensitive thana luminometer, and so the IVIS 50 imaging system (Xenogen, Alameda,Calif.) was used for the work presented here. Luciferase reacts with itssubstrate, luciferin, to produce oxyluciferin and light (11). BecauseATP and oxygen are required for the reaction, photon production has beenused as a quantitative measurement of cellular viability (14). Animalstudies have demonstrated a strong correlation between the abundance ofemitted photons and number of cells present in a given tissue or animal(5,11).

In general, the field of oncology has utilized BLI extensively to studythe effects of anti-cancer therapy in vivo (15,23). However, applicationof BLI to study hematologic malignancies has been limited (6,22,44), andto date, there are no bioluminescent hematologic cell lines commerciallyavailable (Xenogen Corp., Alameda, Calif.). Validation of BLI inpreclinical models has been carried out using currently availablemethods and evidence indicates that BLI has excellent sensitivity andoffers unique advantages (5,25,31,33). For example, non-invasive BLIallows visualization of cells temporally and spatially, thus permittingsmall changes in cell number and localization to be detected over time(24,31). In addition, animals need not be sacrificed at each samplingtime point, decreasing the number of animals that are required for anexperiment and minimizing inconsistency from animal-to-animalvariations.

There is a significant difference between the sensitivity of BLI and thetrypan blue dye exclusion assay. For a cell to be detected as nonviablewith the trypan blue assay, the dye must enter the cytoplasm of thecell. Trypan blue is a relatively large molecule (mw 960.8) and whilemany cells may be metabolically dead, their membranes could besufficiently intact to exclude the dye to appear viable. In contrast,BLI detects killing sooner because ATP is no longer available in ametabolically dead cell. The results are in strong agreement withKuzmits et al. (17) who found that an ATP/bioluminescent assay withHL-60 cells indicated nearly complete killing after a 24 hour incubationwith 5.7 μmol/l doxorubicin, while the trypan blue assay indicatedalmost no killing after 48 hours with the same drug concentration.Furthermore, Petty et al. (30) reported that a bioluminescent ATP assaycould detect as few as 1500 viable cells/well while the MTT assay couldnot detect less than 25,000 cells/well.

Bacterial toxins have been investigated for their anti-cancertherapeutic potential for many years. Several widely-studied toxinsinclude diphtheriae toxin (DT) and Pseudomonas exotoxin A (PE) (16). Toincrease the specificity of these toxins, their toxic domains are oftenfused to other molecules that target the toxin to certain cell types.For example, ONTAK, a recently approved drug used to treat cutaneousT-cell lymphoma, is a fusion molecule of DT and IL-2 (10,26).

As discussed above, the oral bacterium A. actinomycetemcomitans producesa 113 kDa protein toxin, leukotoxin (LtxA), which kills only blood cellsof humans and Old World Primates (3739). Furthermore, and in accordancewith a first aspect of the invention, a strain has been identified whosepurified LtxA does not lyse RBCs. LtxA binds to LFA-1 on host cells (19)and destroys cells by apoptosis or necrosis (18). Because LtxA alreadyhas specificity towards WBCs, it has been proposed that the proteinmight serve as an effective targeted therapy for hematologicmalignancies. In addition, the toxin kills host cells by disruption ofthe cell membrane (18) and therefore represents a mechanism of actionthat is different from other chemotherapeutic agents.

In an effort to further evaluate the therapeutic potential of LtxA, atest was conducted with the present in vivo screening assay.Accordingly, the toxic effects LtxA against HL-60luc cells in blood wereexamined. As set forth and demonstrated herein, the toxin remains highlyactive in human blood and kills HL60luc cells as efficiently as in RPMImedium. In addition, bone marrow progenitor cell proliferation assaysindicate that LtxA is active toward myeloid progenitor cells and has anIC₅₀ value in the low ng/ml range. Preliminary studies also suggest thatLtxA is active mice and does not display toxicity when injected at highdoses into mice.

With the ability to rapidly determine HL-60luc cell viability in thepresence of biological fluids, it is possible to efficiently screenthousands of different compounds at a time for anti-leukemia activity.Assays may be performed in 96- or 384-well dishes in the presence ofphysiologically-relevant samples such as blood, plasma, or hepatocytes.Indeed, an important preclinical screen to study drug biotransformationis performed in the presence of hepatic material, such as human livermicrosomes, human liver cytosol fractions, and hepatocytes (3). HL-60luccells could be used in high-throughput hepatic screens for drugs withanti-leukemia bioactivity.

In addition to using the present assay comprising the HL-60luc cells fordrug discovery, the test system can be used to monitor the condition ofa patient undergoing drug therapy. Accordingly, the condition andbehavior of a known drug or combination of drugs in the presence ofblood samples from different leukemia patients may be measured anddetermined. For example, neutralizing antibody in a patient's bloodagainst a potential drug might allow a clinician to exclude the drugfrom the therapeutic regimen. Excluding an otherwise ineffective drugmight greatly reduce unwanted side effects. Indeed several studies haveshown a correlation between in vitro chemosensitivity of tumor cells andtherapy outcome (34,42). Such correlations could allow the developmentof assay-directed individualized chemotherapy regimens. Thus the assayof the invention can be used in the following ways:

1. Screening novel drugs for anti-leukemia/cancer activity.2. Determine the best drug dosage for a leukemia/cancer patient.3. Determine which drug might be most effective for a leukemia/cancerpatient.

EXAMPLES

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way beconstrued, however, as limiting the broad scope of the invention.

Example 1 Purification of LtxA from the NJ4500 Strain of A.actinomyceterncomitans

The JP2 strain of A. actinomycetemcornitans produces abundant LtxA, butit does not represent a fresh clinical isolate. Here, LtxA was purifiedfrom the clinical isolate NJ4500 of A. actinomycetemcomitans. Thisstrain also produces and secretes a large amount of LtxA, but the cellsadhere to surfaces instead of growing planktonically. This type ofadherent growth results in a relatively low number of cells per volume.The cell density of adherent cells was increased by increasing thesurface area on which the cells can grow through the addition ofspherical glass beads. Soda lime beads provided the greatest amount ofLtxA when compared to Pyrex glass beads. The amount of LtxA that waspurified from NJ4500 in the presence of soda lime beads wasapproximately twice that of JP2.

It is important to note that growth of A. actinomycetemcomitans in thepresence of both types of glass beads was similar suggesting thatdifferences in LtxA quantity was not due variable growth. A.actinomycetemcomitans strains JP2 and NJ4500 are known in the art. Allbacteria were grown in A. actinomycetemcomitans growth medium (AAGM) asknown in the art. Plates were incubated at 37° C. in 10% CO₂ for 4 days.Broth cultures were incubated for 24 h unless otherwise noted.

LtxA was isolated from JP2 by growing cells in 5 ml AAGM broth for 7-9 hand then diluted into 400 ml fresh AAGM broth. These cultures were thengrown for 13-17 h before harvesting supernatant. To obtain supernatant,cultureswere centrifuged at 17,000 g for 10 minutes at 4 8 C. Thesupernatant was filtered through a 0.22 mm low-protein binding membranefilter. For every 100 ml of filtered supernatant, 32.5 g (NH₄)₂SO₄ wasadded. The mixture was gently rocked at 4 8 C for 1 h. The precipitatedprotein was collected by centrifugation at 10,000 g for 20 min at 4 8 C.The pellet from 400 ml supernatant was then resuspended in 2 ml LtxAbuffer (20 mM Tris-HCl, pH 6.8, 250 mM NaCl, and 0.2 mM CaCl₂).

The resuspended pellet was loaded on a column packed with 40 ml ofSephadex G-100 (Sigma, St. Louis, Mo.). Protein was eluted in 1 mlfractions with LtxA buffer. Protein content in each fraction wasdetermined with the Bradford reagent. The three fractions with thehighest protein content were combined, aliquoted and stored at −80° C.The purity of LtxA was determined on a 4-20% SDS-PAGE gel and theconcentration was determined by the BCA assay according to themanufacturer's protocol (Pierce, Rockford, Ill.).

LtxA was purified from the adherent strain NJ4500 by first growing cellsin tubes filled with 5 ml AAGM broth for 14 h and then transferring 20ml of growing cultures into 400 ml AAGM broth in a 500-ml bottle. Priorto adding 400 ml sterile AAGM broth to the 500 ml-bottle, 300 g of glassbeads (or no beads, for controls) were autoclaved inside the bottle. Thesoda lime beads were obtained from Fisher Scientific (cat. 11-312C) andpyrex beads from Corning Incorporated (cat. 7268-5). The inoculatedbottle was grown for 36-40 h as described above. During growth, thebottle was inverted several times to allow adherent cells to coat allthe beads. After growth, the broth was removed and centrifuged andprocessed as described above for JP2 LtxA. For these experiments, cellswere not removed from the beads.

Although adherent variants such as NJ4500 retain a greater amount ofLtxA than the nonadherent variants, a large amount of secreted LtxA fromNJ4500 can still be harvested. Because NJ4500 attaches avidly tosurfaces, the number of growing cells per volume can be increased byadding 5 mm glass beads to the growth medium. In methods using one oftwo different types of glass beads, Pyrex and soda lime, the yield ofLtxA from cells growing on Pyrex was significantly reduced when comparedto the control of no glass beads or soda lime beads.

Example 2 Imaging of Mice Injected with HL-60luc

The images of the mice shown in FIG. 3 were collected using in vivobioluminescence imaging. The SCID mouse model has been used extensivelyfor the study of hematologic malignancies, and the pattern of leukemiadisplayed in SCID mice closely resembles human clinical disease. In themodel, leukemia cells are injected into SCID mice, usuallyintravenously. A commonly used leukemia cell line is HL-60, originallyisolated from a 36-year-old female patient with acute promyelocyticleukemia. Animal studies have shown that HL-60 cells can infiltrate bonemarrow, the spleen, thymus, kidney, liver, lungs, and even the brain. Ithas been reported that the mean survival time for SCID mice that wereinjected with HL-60 cells was 42.5 days following injection; however,this time can vary depending on the passage state of the HL-60 cellsbeing injected.

In vivo bioluminescence imaging (BLI) is a technology that allowsvisualization of live bioluminescent cells (mammalian, bacterial,viruses) in a living animal, without sacrificing the animal. Cells to bevisualized are engineered to express luciferase, which reacts with itssubstrate, to result in light production. Because the reaction alsorequires ATP, bioluminescence has also been used as a measure ofviability. In bacteria, the substrate is encoded within the same operonas the luciferase enzyme. In the mammalian system, the substrate,luciferin, must be injected separately into the animal for thelight-producing reaction to take place. Visualization of luminescentcells requires a highly-sensitive CCD camera that can detect low-levellight within a short period of time. We currently maintain the XenogenIVIS 50 imaging system for this purpose (Xenogen Corp., Alameda,Calif.). The distribution and abundance of luciferase-producing cellscan be quantified by anesthetizing the animals and imaging them with theIVIS 50 imaging system.

BLI allows visualization of cells temporally and spatially, thusallowing small changes in cell number and localization to be detectedover time. In contrast, using standard methods, animals must besacrificed and extensive histological examination performed to localizecells in question. In general, the field of oncology has utilized BLIextensively, however, application of BLI to study hematologicmalignancies has been limited, and to date, there are no bioluminescenthematologic cell lines commercially available (Xenogen Corp., Alameda,Calif.).

In the oncology models, engineered malignant cells are injected intoanimals and the progression of cancer is observed. In addition,transgenic light-producing mice are available for use in severaloncology models (Xenogen Corp., Alameda, Calif.). Metastasis andregression can be monitored with great sensitivity and efficiency withthe IVIS instrument. Of great significance, the effects of anticancertherapy can be determined before the endpoint of death is reached.

Example 3 Prepareation of Assay System Experimental Cells and GrowthConditions

HL-60 cells were obtained from American Type Culture Collection (ATCC)and maintained in RPMI+10% fetal bovine serum (FBS) (Invitrogen,Carlsbad, Calif.) at 37° C.+5% CO₂. Escherichia coli was grown in LBmedium at 37° C. A. actinomycetemcomitans strains were grown in AAGM at37° C.+10% CO₂ as previously described (12).

DNA Manipulations

The luciferase-encoding plasmid for transfecting HL-60 cells wasconstructed by cloning luciferase gene from pGL3 (Promega, Madison,Wis.) into the geneticin resistance gene-containing plasmid pCI-neo(Promega, Madison, Wis.). Both plasmids were digested with BglII andXbaI and the Neo-containing fragment was then ligated to the pGL3fragment that contained the luciferase gene. The mixture was transformedinto E. coli and the bacteria were selected on LB+carbenecillin (50μg/ml). Plasmid from bacteria was prepared using the plasmid miniprepkit (Qiagen, Valencia, Calif.). The new plasmid, encoding bothluciferase and geneticin, was designated pMP1.

The plasmid, pMP1, was transfected into HL-60 cells by electroporation.Briefly, 106 cells were resuspended in 400 μl electroporation buffer (20mM HEPES pH 7.0, 137 mM NaCl, 5 mM KCl, 0.7 mM Na₂HPO₄, 6 mM glucose,0.1 mM β-mercaptoethanol). Plasmid pMP1 was added at a concentration of12.5 μg/ml and the mixture was incubated for 5 minutes on ice. Themixture was added to a cuvette and a pulse of 380 V was administered.Five ml of fresh RPMI medium was added to the cells and they were grownfor 24 hours before geneticin was added.

Preparation of Cytotoxic Agents

Bacterial leukotoxin (LtxA) was purified from A. actinomycetemcomitansas previously described (7). LtxA was stored in 100 μl aliquots at −80°C. until used. A stock solution of chlorambucil (Sigma, St. Louis, Mo.)was prepared by dissolving 30 mg into 1 ml of DMSO. The drug was freshlyprepared prior to each experiment.

Bioluminescent Imaging (BLI)

For detection of bioluminescence (BL) from cultured HL-60luc cells, 200μl of cells were mixed with 1 μl luciferin (15 mg/ml) and then imagedwith the IVIS 50 imaging system (Xenogen Corp., Alameda, Calif.). Foranimal studies, Swiss Webster mice were first injected with 106 HL-60luccells (resuspended in PBS) or PBS control intraperitoneally (i.p.) orintravenously (i.v.). Mice were then anesthesized with acepromazine (0.3mg/40 g, i.p.) and a rodent cocktail [ketamine (20 mg/ml) and xylazine(2.5 mg/ml)] (0.1 ml/25 g, i.p.). Luciferin was then injected (150mg/kg) i.p and the mice were imaged with the IVIS 50 instrument atdifferent times. Images were analyzed using the Living Image Software(Xenogen Corp., Alameda, Calif.).

Results Construction of a Stable HL-60 Luciferase-Expressing Cell Line

To generate an HL-60 cell line that stably expresses luciferase, aplasmid was constructed by cloning the luciferase gene from pGL3 intothe geneticin resistance gene-containing plasmid pCI-neo. The modifiedplasmid, pMP1, was then electroporated into HL-60 cells (obtained fromATCC) and grown under geneticin selection. When geneticin was includedin the growth medium to select for the plasmid, bioluminescence (BL) wasobserved, indicating that cells received the luciferase-encodingplasmid. FIG. 8A shows HL-60 cells that were transfected with pMP1 andthen grown in wells with different concentrations of geneticin.Bioluminescence was detected with the IVIS 50 instrument. Cells weregrown for 8 weeks longer to allow the generation of stable clones. After8 weeks, geneticin selection was removed to determine if theluciferase-encoding gene had successfully integrated into the genome.Even after growing cells for many generations without selection, theHL-60 cells still emitted light, suggesting that stable transfectantshad been obtained.

To continue studies, a homogeneous population of cells derived from asingle stable clone was isolated by performing minimal dilutions withstable transfectants. Cells were diluted to approximately one cell/wellin a 96-well dish and then examined microscopically to exclude wellsthat received more than one cell. Dishes were further incubated and thenimaged with the IVIS 50 instrument. Cells were transferred to largerdishes, grown and then and saved in liquid nitrogen. Viability of thesesaved cells was greater than 90%.

An important property for BLI studies is photon flux per cell(photons/second/cell). The flux/cell for one specific clone that wasused in all subsequent assays described here was calculated. Thecalculated value of 16 photons/second/cell is consistent with valuesobtained from other engineered cell lines (Xenogen Corp., Alameda,Calif.). It is believed that this is the first HL-60 cell line that hasbeen engineered to stably express luciferase.

To confirm that the engineered HL-60 cells maintain basic growthcharacteristics, growth studies were performed comparing HL-60luc cellsto parental HL-60 cells. Cells were grown in RPMI with 10% FBS and thencounted with a Vi-CELL cell viability analyzer (Beckman Coulter, Inc.,Miami, Fla.). Growth curve experiments in RPMI for the two cell linesindicated that HL-60luc cells behave like the parental cell line. FIG.8B shows growth curves for parental HL-60 and engineered HL-60luc cells.Cells were grown in RPMI as described and viable cells were counted withthe ViCELL cell counter.

Detection of HL-60luc in Blood

To determine the kinetics of bioluminescence in blood, 6×105 HL-60luccells were mixed with human peripheral blood and luciferin was added tothe mixture. BL was then measured over time as photons/second from eachsample. FIG. 9A shows the kinetics of BL over time. HL-60luc cells weremixed with blood and luciferin and then imaged with the IVIS 50instrument at the indicated time points. The observed pattern was highlyreproducible. The signal peaked at one hour and was approximately 200times greater than the background signal from blood alone. FIG. 9A.These results were highly reproducible and a similar pattern wasobtained when the same experiment was performed in RPMI. BL values inRPMI were approximately two-fold greater than in blood likely due tolight absorption by the blood.

The sensitivity of detection in blood was then determined. Differentnumbers of HL-60luc cells were mixed with blood (200 μl total) andluciferin was added to each sample. The mixtures were incubated at 37°C. for one hour and BL was measured. FIG. 9B shows detection limit ofHL-60luc cells. Cells were mixed with blood and luciferin and thenincubated for one hour before imaging. Approximately 1000 cells could bedetected above the background level of the blood alone. The signalemitted from the highest number of cells tested (1.25×106) wasapproximately 2000 times greater than blood alone. The BL signalcorrelated strongly with cell number. FIG. 9C shows that the number ofHL-60luc cells shows a linear correlation with BL.

Sensitivity of HL-60luc Cells to a Bacterial Toxin

The gram negative bacterium, A. actinomycetemcomitans, producesleukotoxin (LtxA), a protein toxin that kills specifically white bloodcells from humans and Old World Primates (37-39) and red blood cells(1). Examination of LtxA from a strain of A. actinomycetemcomitans,NJ4500, revealed that this purified protein does not lyse erythrocytesin vitro compared to LtxA from the standard strain, JP2. FIG. 10A showsthe lysis of human red blood cells by LtxA from two different strains ofA. actinomycetemcomitans. Because erythrocyte lysis would be anundesirable property for a chemotherapeutic agent, studies here employLtxA from NJ4500.

To determine if HL-60luc cells are equally sensitive to LtxA as parentalHL-60 cells, cell killing was assayed by LtxA in RPMI. HL-60 cells weremixed with LtxA and viability was measured with the trypan blue dyeexclusion assay using the Vi-CELL instrument. LtxA had an equal toxiceffect on both cell lines. FIG. 10B shows that HL-60 and HL-60luc cellsare equally sensitive to killing by LtxA from strain NJ4500. Assays wereperformed in RPMI medium and viability was determined using the trypanblue dye exclusion assay. This result was highly reproducible. Thus, theHL-60luc cell line is similar to the parental HL-60 cell line for itsensitivity to a bacterial toxin.

LtxA Activity in Whole Blood

To determine if LtxA is active in whole blood and retains its ability tokill HL-60 cells, HL-60luc cells were resuspended in blood or RPMI anddifferent concentrations of purified LtxA or LtxA buffer was added tothe HL-6luc-blood mixture and incubated at 37° C. for 4 hours. BLI wasthen measured and relative viabilities were determined comparingexperimental values to the buffer-containing sample. LtxA was highlyactive in whole blood against HL-60luc cells and this activity wassimilar to that seen in RPMI. FIG. 11A shows the activity of LtxAagainst HL60luc cells in whole human blood and RPMI medium. Viabilitywas measured using BL.

The sensitivity of BL to trypan blue as an assay for cell viability wasalso compared. Luminescence was significantly more sensitive than trypanblue. FIG. 11B shows the comparison between BL and trypan blue asviability assays for LtxA-mediated cytotoxicity. Cells were incubated inRPMI medium with LtxA or buffer for 4 hours and viability wasdetermined. Nearly complete cell killing was observed with leukotoxinconcentrations as low as 10 ng/ml using BL values. In contrast, trypanblue revealed that only 35% killing had occurred at this concentration.

To determine if the difference in detection limit between the twomethods was specific for LtxA-mediated cytotoxicity, another compound,chlorambucil, was used to induce cell death. Chlorambucil alkylates DNAand induces apoptosis (2,21) and therefore represents a mechanism ofkilling different from that of LtxA. For chlorambucil, it was alsoobserved that BLI was a more sensitive assay than trypan blue fordetecting viability. FIG. 4C shows the comparison between BL and trypanblue as viability assays for chlorambucil-mediated cytotoxicity. Cellswere incubated in RPMI medium with chlorambucil or buffer for 24 hoursand viability was determined. At a chlorambucil concentration of 0.03mg/ml, BLI revealed approximately 90% cell death after 24 hours whiletrypan blue revealed essentially no killing (FIG. 11C).

Visualization of HL-60luc in Mice

Mouse models for human leukemia utilize HL-60 cells that are injectedeither i.p. (28) or i.v. (40,41). To determine if the HL-60luc cellscould be visualized in living mice, approximately 106 HL-60luc cellswere injected i.p. or tail i.v. FIG. 12 shows Swiss-Webster mice thatwere anesthesized with XXX and injected with 106 HL-60luc cellsintraperitoneally (i.p.; top) or intravenously (i.v.; bottom) andfollowed by luciferin i.p. Mice were imaged with the IVIS 50 instrumentat different times post-luciferin injection. The scale on the right ofeach image indicates surface radiance (photons/second/cm2/steradian).Luciferin was administered immediately following injection of cells andthe animals were imaged with the IVIS 50 instrument. The cells could bedetected with a 2-3 minute exposure when administered by either route.The signal was greatest for i.p.-injected cells immediately followinginjection while the signal for i.v.-injected cells peaked approximately35 minutes post luciferin (FIG. 12). Interestingly, the signal observedfor i.v. injection follows the path of the tail vein and then dissipatesas the cells become diluted through other blood vessels. Thus, HL-60cells can be visualized in a living animal at concentrations normallyused for the SCID mouse model for human leukemia.

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All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

While preferred embodiments of the invention have been shown anddescribed herein, it will be understood that such embodiments areprovided by way of example only. Numerous variations, changes andsubstitutions will occur to those skilled in the art without departingfrom the spirit of the invention. Accordingly, it is intended that theappended claims cover all such variations as fall within the spirit andscope of the invention.

1. A composition comprising leukotoxin proteins isolated from abacterium, wherein greater than 85% of the leukotoxin proteins arechemically modified at a basic amino acid residue.
 2. The composition ofclaim 1 wherein the bacterium is Actinobacillus actinomycetemcomitans.3. The composition of claim 1 wherein the bacterium is a NJ4500 strainof Actinobacillus actinomycetemcomitans.
 4. The composition of claim 1wherein the proteins induce cell death in myeloid leukocytes.
 5. Thecomposition of claim 1 wherein the protein induces cell death in anyoneof white blood cells, neutrophils, monocytes, basophils, andeosinophils.
 6. The composition of claim 1 that is substantiallynon-toxic to lymphoid leukocytes.
 7. The composition of claim 6 that issubstantially non-toxic to lymphocytes.
 8. The composition of claim 1that is substantially non-toxic to red blood cells.
 9. The compositionof claim 1 wherein the chemical modification is a fatty acidmodification.
 10. The composition of claim 1 wherein the basic aminoacid residue is a lysine.
 11. The composition of claim 1 wherein greaterthan 90% of the leukotoxin proteins are chemically modified at a basicamino acid residue.
 12. A composition comprising leukotoxin proteins,wherein 85% of the leukotoxin proteins have a pI less than 9.0.
 13. Thecomposition of claim 12, wherein 90% of the leukotoxin proteins have apI less than 9.0.
 14. The composition of claim 13, wherein 95% of theleukotoxin proteins have a pI less than 9.0.
 15. The composition ofclaim 14, wherein 100% of the leukotoxin proteins have a pI less than9.0
 16. A composition comprising leukotoxin proteins, wherein 85% of theleukotoxin proteins have a pI less than 8.5.
 17. The composition ofclaim 16, wherein 90% of the leukotoxin proteins have a pI less than8.5.
 18. The composition of claim 17, wherein 95% of the leukotoxinproteins have a pI less than 8.5.
 19. The composition of claim 18,wherein 100% of the leukotoxin proteins have a pI less than 8.5.
 20. Thecompositions of claim 12 wherein the leukotoxin proteins are isolatedfrom Actinobacillus actinomycetemcomitans.